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SnapShot-Seq: a method for extracting genome-wide, in vivo mRNA dynamics from a single total RNA sample.

Gray JM, Harmin DA, Boswell SA, Cloonan N, Mullen TE, Ling JJ, Miller N, Kuersten S, Ma YC, McCarroll SA, Grimmond SM, Springer M - PLoS ONE (2014)

Bottom Line: SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA.We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors.SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment.

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SnapShot-Seq-derived timescales for ten human tissues and technical controls.(A) Lifetimes obtained from total RNA-Seq performed on ten human tissues, using the SOLiD Whole Transcriptome Sequencing method [23] with RiboMinus rRNA-depletion. T5, T3, and Tγ are as defined as in Fig. 1. (B) A comparison of lifetimes across different sequencing methodologies. We performed sequencing on SOLiD (S) or Illumina (I); hybridization-based rRNA depletion with RiboMinus (M) or RiboZero (Z); and compared RNA samples isolated and prepared into libraries on several different days. We performed Illumina total RNA-Seq using the dUTP method [19]. Error bars indicate 95% confidence from Monte Carlo simulations from individual biological samples.
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pone-0089673-g002: SnapShot-Seq-derived timescales for ten human tissues and technical controls.(A) Lifetimes obtained from total RNA-Seq performed on ten human tissues, using the SOLiD Whole Transcriptome Sequencing method [23] with RiboMinus rRNA-depletion. T5, T3, and Tγ are as defined as in Fig. 1. (B) A comparison of lifetimes across different sequencing methodologies. We performed sequencing on SOLiD (S) or Illumina (I); hybridization-based rRNA depletion with RiboMinus (M) or RiboZero (Z); and compared RNA samples isolated and prepared into libraries on several different days. We performed Illumina total RNA-Seq using the dUTP method [19]. Error bars indicate 95% confidence from Monte Carlo simulations from individual biological samples.

Mentions: We used our model to determine mRNA processing times (T5, T3, and Tγ, as defined in Fig. 1C) for ten human tissues after performing strand-specific total RNA-Seq [23] from ribosomal RNA-depleted total RNA isolated from each tissue (Fig. 2A). Using a Monte Carlo approach, we repeatedly randomly sampled sets of five genes and solved for the RNA processing times (Materials and Methods), which produced a distribution for each processing time. Across ten human tissues, we find lariat formation (T5) takes an average of 1–2 minutes, exon ligation (T3) takes an average of 30–70 seconds, and intron degradation (Tγ) takes an average of 20–30 seconds. These results are consistent with the results of complementary techniques that have examined specific steps in the mRNA lifecycle at small numbers of introns or genes [8], [11], [24]–[26]. We also used our model to calculate mRNA lifetimes (Tμ). Across the same ten human tissues, the average mRNA lifetime varied from just under 1 hour to nearly 4 hours (Table S1 in File S1). These values are similar to the ∼5 hour average mRNA lifetime found by previous studies in mouse and human samples [27].


SnapShot-Seq: a method for extracting genome-wide, in vivo mRNA dynamics from a single total RNA sample.

Gray JM, Harmin DA, Boswell SA, Cloonan N, Mullen TE, Ling JJ, Miller N, Kuersten S, Ma YC, McCarroll SA, Grimmond SM, Springer M - PLoS ONE (2014)

SnapShot-Seq-derived timescales for ten human tissues and technical controls.(A) Lifetimes obtained from total RNA-Seq performed on ten human tissues, using the SOLiD Whole Transcriptome Sequencing method [23] with RiboMinus rRNA-depletion. T5, T3, and Tγ are as defined as in Fig. 1. (B) A comparison of lifetimes across different sequencing methodologies. We performed sequencing on SOLiD (S) or Illumina (I); hybridization-based rRNA depletion with RiboMinus (M) or RiboZero (Z); and compared RNA samples isolated and prepared into libraries on several different days. We performed Illumina total RNA-Seq using the dUTP method [19]. Error bars indicate 95% confidence from Monte Carlo simulations from individual biological samples.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3935918&req=5

pone-0089673-g002: SnapShot-Seq-derived timescales for ten human tissues and technical controls.(A) Lifetimes obtained from total RNA-Seq performed on ten human tissues, using the SOLiD Whole Transcriptome Sequencing method [23] with RiboMinus rRNA-depletion. T5, T3, and Tγ are as defined as in Fig. 1. (B) A comparison of lifetimes across different sequencing methodologies. We performed sequencing on SOLiD (S) or Illumina (I); hybridization-based rRNA depletion with RiboMinus (M) or RiboZero (Z); and compared RNA samples isolated and prepared into libraries on several different days. We performed Illumina total RNA-Seq using the dUTP method [19]. Error bars indicate 95% confidence from Monte Carlo simulations from individual biological samples.
Mentions: We used our model to determine mRNA processing times (T5, T3, and Tγ, as defined in Fig. 1C) for ten human tissues after performing strand-specific total RNA-Seq [23] from ribosomal RNA-depleted total RNA isolated from each tissue (Fig. 2A). Using a Monte Carlo approach, we repeatedly randomly sampled sets of five genes and solved for the RNA processing times (Materials and Methods), which produced a distribution for each processing time. Across ten human tissues, we find lariat formation (T5) takes an average of 1–2 minutes, exon ligation (T3) takes an average of 30–70 seconds, and intron degradation (Tγ) takes an average of 20–30 seconds. These results are consistent with the results of complementary techniques that have examined specific steps in the mRNA lifecycle at small numbers of introns or genes [8], [11], [24]–[26]. We also used our model to calculate mRNA lifetimes (Tμ). Across the same ten human tissues, the average mRNA lifetime varied from just under 1 hour to nearly 4 hours (Table S1 in File S1). These values are similar to the ∼5 hour average mRNA lifetime found by previous studies in mouse and human samples [27].

Bottom Line: SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA.We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors.SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment.

Show MeSH
Related in: MedlinePlus